AMPK activation enhances osteoblast differentiation on a titanium disc via autophagy

Purpose The acquisition of osseointegration during implant therapy is slower and poorer in patients with diabetes compared with healthy persons. The serum concentration of adiponectin in patients with type II diabetes is lower than that of healthy persons via the suppression of AMP-activated protein kinase (AMPK). Therefore, we hypothesized that the AMPK activation enhances bone formation around implants, resulting in the improved acquisition of osseointegration. The purpose of this study was to evaluate the impact of AMPK activation on osteoblast differentiation and its mechanism of downstream signaling on titanium disc (Ti). Methods Confluent mouse pre-osteoblasts (MC3T3-E1) cells (1 × 105 cells/well) were cultured with BMP-2 for osteoblast differentiation, in the presence or absence AICAR, an AMPK activator. We examined the effects of AMPK activation on osteoblast differentiation and the underlying mechanism on a Ti using a CCK8 assay, a luciferase assay, quantitative RT-PCR, and western blotting. Results Although the proliferation rate of osteoblasts was not different between a Ti and a tissue culture polystyrene dish, the addition of AICAR, AMPK activator slightly enhanced osteoblast proliferation on the Ti. AICAR enhanced the BMP-2-dependent transcriptional activity on the Ti, leading to upregulation in the expression of osteogenesis-associated molecules. AICAR simultaneously upregulated the expression of autophagy-associated molecules on the Ti, especially LC3-II. AdipoRon, an adiponectin receptor type1/type2 activator activated AMPK, and upregulated osteogenesis-associated molecules on Ti. Conclusions AMPK activation enhances osteoblast differentiation on a Ti via autophagy, suggesting that it promotes the acquisition of osseointegration during implant therapy. Supplementary Information The online version contains supplementary material available at 10.1186/s40729-024-00525-2.


Background
AMPK has been reported to be a conserved sensor of cell energy conditions [1].AMPK has previously been reported to be activated by metabolic stresses and to switch on catabolic pathways while switching off biosynthetic pathways to maintain the intracellular energy equilibrium [2].AMPK activation promotes cell viability and mitochondrial biogenesis and fission, as well as the expression of antioxidant enzymes [3].There are some reports that AMPK has been reported to stimulate osteoblast differentiation and mineralization in preosteoblast cell lines on tissue culture polystyrene dishes (TCPD) using osteogenesis induced medium (OIM) [4,5].Furthermore, AICAR, an AMPK activator stimulated the autophagy signaling pathway in osteoblast lineages including mesenchymal stem/stromal cells on TCPD [6,7].We recently reported that AICAR upregulated osteoblastic mineralization on titanium disc (Ti) [8], but not TCPD.However, most of these reports were obtained from osteoblasts cultured on TCPD using osteogenesis induced medium (OIM), and few have been investigated those of titanium.Furthermore, no reports have verified autophagy-regulated osteoblast differentiation on titanium, but not on TCPD.
Adiponectin is a fat-derived adipokine that plays an important role in controlling the balance of energy metabolism at a systematic level via AMPK activation [9,10].The concentration of adiponectin in the serum is significantly reduced in patients with type 2 diabetes, resulting in diabetes-induced bone loss [11].Therefore, AMPK, adiponectin downstream signaling has been proposed to be a potential therapeutic target to promote osseointegration in bone-to-implant contact and osseointegration, especially in patients with type 2 diabetes [12].
In the present experiments, we hypothesize that the addition of AMPK activator enhances osteoblast differentiation in the around peri-implant bone, leading to the promotion of earlier and stronger bone-to-implant contact and osseointegration, even though diabetes.We examined the effects of AMPK activation on the expression of osteoblast differentiation-associated molecules on a Ti in mouse pre-osteoblasts, as well as its underlying mechanism.
The purpose of this study was to evaluate the impact of AMPK activation on osteoblast differentiation and its mechanism of downstream signaling on titanium discs.

Surface topography of Ti
Titanium surface roughness was evaluated parameter the arithmetic mean height of the surface (Sa) using a fiber laser (MD-F3000, Keyence, Osaka, Japan: Fig. 1A), with a 50 × 50 mm filter in three different areas on each sample.Characterization of material surface and condition of cells on titanium.MC3T3E1 cells cultured on titanium disc at 24 h.Cells were fixed with 10% formalin, dehydrated in grated concentrations of ethanol, and dried by liquefied CO 2 using a critical point dryer.Samples were sputtered with gold for 2 min and observed by scanning electron microscope (JSM-6330F, JEOL, Tokyo, Japan).

CCK-8 proliferation assay
Cellular proliferation was measured using a counting Kit-8 assay (CCK-8; Dojindo Laboratories, Kumamoto, Japan).Briefly, the cells were seeded in 24-well plates at a density of 1.0 × 10 4 cells/mL in 500 μL of MEM-α.After each cultivation, the CCK-8 reagent (10 μL) was added to each well and the plates were incubated at 37 °C for 1 h.After the collection of conditional culture medium (100 μL) from each well, absorbance was read at 450 nm using a microplate reader (Mode 680; Bio-Rad).Culture solution containing the CCK-8 reagent but without cells was used as a blank control.

Calcein staining
Cells (10 × 10 4 cells/mL) were plated on a titanium disc inserted in a 24-well plate (500 μL/well).After cultivation for 7 days, cells on the titanium disc were stained with calcein-AM solution (1 µL/mL, DOJINDO).The number of cells on the titanium disc was observed under a fluorescence microscope.

Transcriptional activity assay
For promoter assays, the Id promoter was cloned into the pGL4 basic luciferase plasmid (Promega; Madison, WI, USA), and the resulting construct was transfected into MC3T3-E1 cells using Lipofectamine 3000 (Thermo Fisher, CA, USA).Luciferase activity was measured using a luciferase reporter assay system (Promega).These cells were cotransfected with the pCMVgalactosidase reporter plasmid (Promega) and the transfection activity was normalized by measuring the galactosidase activity.

RNA isolation and quantitative reverse transcription polymerase chain reaction
Total RNA was extracted from the cells using the TRIzol reagent.First-strand complementary deoxyribonucleic acid (cDNA) was synthesized from 3 µg of total RNA using ReverTra Ace reverse transcriptase according to the manufacturer's instructions (TOYOBO CO., LTD, Osaka, Japan).To detect mRNA expression, we selected specific primers based on the nucleotide sequence of the resultant cDNA (Additional File 1: Table S1).The relative messenger RNA (mRNA) expression was normalized as the ratio of the expression level of the targeted mRNAs to that of β-actin.

Western blot analysis
Cells were lysed in TNT buffer [0.1 M Tris-HCl (pH, 7.5), 0.15 M NaCl, 0.05% Tween-20; Roche, Basel, Switzerland].The protein content was measured using a protein assay kit (Thermo Fisher, CA, USA).Subsequently, 20 µg of each protein were subjected to 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated proteins were transferred electrophoretically onto a polyvinylidene fluoride membrane at 80 V and 4 °C for 1.5 h.The membrane was incubated with antibodies against Runx2, Osterix/Sp7 (OSX; Abcam, Cambridge, UK), p-Smad1/5/9, Smad1, P-AMPK, AMPK, Beclin-1, LC3B, p-ULK1, ULK-1, BNIP-3 (Cell Signaling Technology, Tokyo, Japan), and β-actin (Sigma-Aldrich Co.) diluted (1:1000) in 5% BSA and Tris-buffered saline with Tween-20 (TBST; 10 mM Tris-HCl, 50 mM NaCl, 0.25% Tween-20) plus 0.01% azide overnight at 4 °C.The blots were washed in TBST and incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin-G secondary antibodies.The antibodies were diluted (1:2000) in 5% skimmed milk in TBST and Fig. 1 AICAR slightly upregulated the BMP-2-induced proliferation rate in osteoblasts on a titanium disc.A Characterization of material surface of titanium.Surface topology of Ti using laser fiber scanning and those in without or with MC3T3-E1 cells cultured after 24 h using scanning electron microscope.B MC3T3-E1 cells were treated with BMP-2 (50 ng/mL) in the presence or absence of AICAR (500 μM) and stained on a titanium disc using calcein-AM.The number inserted at the bottom left of each photo indicates the mean from the positive cells on three Ti discs (mean ± SEM).C Cells treated with BMP-2 (50 ng/mL) were cultured with or without AICAR (500 μM) on the titanium disc, and the rate of cell proliferation as determined using a CCK-8 assay.The bar graphs show the mean from five culture wells (mean ± SEM).**Indicates P < 0.01 vs. treatment with BMP-2 alone were developed using an enhanced chemiluminescent system (GE Healthcare, Tokyo, Japan).

Statistical analyses
Data are expressed as the mean ± standard error of the mean.Differences were analyzed by one-way analysis of variance and Scheffe's multiple comparison test.P values of < 0.05 were considered significant.

AICAR slightly upregulated the proliferation rate of osteoblasts on a titanium disc (Ti)
To clarify the attachment and viability of MC3T3-E1 cells on a titanium disc (Ti), we first examined the cellular morphology and proliferation on Ti using calcein staining and a CCK-8 proliferation assay (Fig. 1B).MC3T3-E1 cells were less attached on Ti compared with TCPD at day 0.However, BMP-2 (50 ng/mL) enhanced the proliferation of the cells on Ti in a time-dependent manner (Fig. 1C).In turn, BMP-2 (50 ng/mL) in the presence of AICAR (500 μM), which is an AMPK activator, slightly upregulated cell proliferation on Ti in a time-dependent manner compared with BMP-2-induced proliferation (Fig. 1C).The number of proliferating cells in the presence of AICAR remained constant over 5 days or was slightly decreased from day 5 after BMP-2 treatment, resulting in a small effect on proliferation in the presence or absence of AICAR.

AICAR enhanced BMP-2-dependent transcriptional activity on Ti with expression of osteoblast differentiation-related molecules
To clarify whether AICAR regulated osteoblast differentiation in MC3T3-E1 cells on Ti, we examined the impact of AICAR on transcriptional activity using a luciferase reporter assay.BMP-2 transiently and greatly increased the transcriptional activity of Id, a BMP-2 downstream signaling gene, in MC3T3-E1 cells on Ti (Fig. 2A).The  2 AICAR enhanced the BMP-2-dependent transcriptional activity on Ti via the expression of osteoblast differentiation-related molecules.A Id promoter site was fused to luciferase and transfected into MC3T3-E1 cells using Lipofectamine 3000.The cells were treated with BMP-2 (50 ng/ mL) with or without AICAR (500 μM) for the indicated times during culture, and luciferase activity was assessed as a measure of Id transcriptional activity.The data are the mean from four culture wells (mean ± SEM).**Indicates P < 0.01 vs. treatment with BMP-2 alone.B-D MC3T3-E1 cells were incubated with BMP-2 in the presence or absence of AICAR (500 μM).Expression of the osteoblast differentiation-related genes Runx2 (Runx2), Osx/Sp7 (OSX/Sp7), and Ocn (Osteocalcin) in MC3T3-E1 cells after incubation with BMP-2 (50 ng/mL).Samples were analyzed by quantitative reverse transcription polymerase chain reaction and were normalized to the ß-actin mRNA.The data are the mean from six culture wells (mean ± SEM).**Indicates P < 0.01 vs. treatment with BMP-2 alone.E Cells were treated with BMP-2 (50 ng/mL) in the presence or absence of AICAR (500 μM).Western blotting was conducted using targeted and ß-actin antibodies.Similar results were obtained in four independent experiments increased transcriptional activity of Id reached a peak at 6 h and declined at 24 h after BMP-2 treatment.Furthermore, the addition of AICAR continuously increased the transcriptional activity, with a peak observed at 12 h in cells on Ti.Furthermore, BMP-2 also induced the expression of the Runx2, OSX/Sp7, and Ocn genes, which are osteoblast differentiation markers, in a time-dependent manner (Fig. 2B-D).Moreover, AICAR addition upregulated the expression of these genes in a time-dependent manner.The increase in the expression of targeted proteins in the presence or absence of AICAR was further confirmed using a western blot analysis (Fig. 2E; Additional File 1: Fig. S1), which yielded similar results, showing that AICAR administration for more than 24 h upregulated the expression of Runx2, OSX/Sp7, and p-AMPK in MC3T3-E1 cells compared with treatment with BMP-2 alone.Similar results were obtained regarding the expression of OIM-induced proteins in MC3T3-E1 cells on Ti using western blot analyses (Additional file 1: Fig. S2).On contrast, AICAR monotreatment slightly upregulated the expression of osteoblast differentiation-related molecules compared with AICAR in presence of OIM (Additional File 1: Fig. S2).

AICAR simultaneously enhanced the expression of BMP-2-induced autophagy-associated molecules in MC3T3-E1 cells on Ti
Osteoblast differentiation has been reported (including our studies) to be regulated by autophagy [13,14].To clarify whether AMPK activation enhances osteoblast differentiation in MC3T3-E1 cells on Ti, we examined the effects of AICAR on the osteogenesis of MC3T3-E1 cells on Ti treated with BMP-2.BMP-2 induced the expression of autophagy-associated genes, such as Ulk1, Becn1, Map1Lc-3, and Parkin, in MC3T3-E1 cells on Ti in a time-dependent manner, with AICAR addition upregulating their expression (Fig. 3A-D).Similar results were obtained regarding the expression of their target proteins, with BMP-2 gradually increasing the conversion of LC3-I to autophagosome-associated LC3-II in MC3T3-E1 cells on Ti (Fig. 3E; Additional File 1: Fig. S3).Furthermore, AICAR addition for more than 12 h Beclin-1 The samples were analyzed by quantitative reverse transcription polymerase chain reaction and were normalized to the ß-actin mRNA.The data are the mean from six culture wells (mean ± SEM).**Indicates P < 0.01 vs. treatment with BMP-2 alone.E Cells were treated with BMP-2 (25 ng/mL) in the presence or absence of AICAR (500 μM).Western blotting was conducted using targeted and ß-actin antibodies.Similar results were obtained in three independent experiments upregulated the expression of their autophagy-associated molecules, together with an increase in the expression of phosphorylated AMPK, especially LC3-II, in MC3T3-E1 cells on Ti.The conversion changes in LC3-II were correlated with the increase in Beclin-1, which is an autophagy marker, in MC3T3-E1 cells.Similar results were obtained regarding the expression of OIM-induced proteins in MC3T3-E1 cells on Ti (Additional file 1: Fig. S2E).On contrast, AICAR monotreatment slightly upregulated the expression of autophagy-related molecules compared with AICAR in presence of OIM (Additional File 1: Fig. S4).
To confirm whether the autophagy-associated molecules regulated osteogenesis, we investigated the inhibitory effect of 3-methhyladnine (3-MA), which is an autophagy inhibitor, on the upregulation of osteoblast differentiation (Fig. 4A; Additional File 1: Fig. S5).
Although autophagy was assessed in the early and late stages of the process, we examined whether autophagy participated in osteoblast differentiation in the early stage using 3-MA, which is an inhibitor of the early stage.Treatment with 3-MA suppressed the expression of Beclin-1 and p-ULK1 in MC3T3-E1 cells on Ti.This treatment simultaneously suppressed osteoblast differentiation markers, such as Runx2 and OSX (Fig. 4B and C).Furthermore, AICAR addition attenuated the suppression of the expression of Runx2 and OSX in MC3T3-E1 cells on Ti, indicating that osteoblast differentiation was regulated by the activation of autophagy, especially in the early stage (Fig. 4D; Additional File 1: Fig. S6).

AdipoRon, an adiponectin receptor activator, also upregulated osteogenesis-associated molecules
Adiponectin activates AMPK via adiponectin receptors in various types of cells, such as microglia, primary rat osteoblasts, and breast cancer cells [15][16][17].In contrast, the decrease in the serum concentration of adiponectin led to the suppression of glucose uptake     4 3-MA suppressed BMP-2-induced osteoblast differentiation molecules and autophagy-related molecules.A BMP-2 (25 ng/mL)-induced osteoblast differentiation molecules were mediated by autophagy signaling.The cells were incubated with BMP-2 in the presence or absence of 3-MA (5 mM), which is a PI3K inhibitor.B, C The bar graphs show the mean from five samples (mean ± SEM).* and **Indicate P < 0.05 and P < 0.01, respectively, vs. treatment with BMP-2 alone.D Cells were treated with BMP-2 (25 ng/mL) plus AICAR (500 μM) in the presence or absence of 3-MA (5 mM).Western blotting was conducted using targeted and ß-actin antibodies.Similar results were obtained in three independent experiments the liver and muscles via the inhibition of AMPK in patients with diabetes [18,19].Therefore, to clarify whether adiponectin activates osteoblast differentiation in Ti, we examined the impact of AdipoRon, which is an adiponectin receptor type1/type2 activator, on transcriptional activity and the expression of osteoblast differentiation-associated molecules in MC3T3-E1 cells on Ti in the presence or absence of AICAR.AdipoRon (50 µM) gradually increased the transcriptional activity of Id (Fig. 5A).The addition of AICAR greatly increased the transcriptional activity.AdipoRon induced the expression of the Runx2, OSX/Sp7, and Ocn genes in MC3T3-E1 cells on Ti in a time-dependent manner (Fig. 5B).Furthermore, AdipoRon induced the expression of autophagy-associated molecules.AICAR addition provided upregulation for more than 24 h compared with treatment with BMP-2 alone (Fig. 5C; Additional File 1: Fig. S7).

Discussion
AMPK activation elicited by AICAR promotes the phosphorylation of Smad 1/5/8 in MC3T3-E1 cells, leading to osteoblast differentiation on tissue culture polystyrene dishes (TCPD) [20].In contrast, BMP-2 phosphorylates Smad1/5, resulting in osteoblast differentiation [21].In the present experiments, BMP-2 induced the proliferation ratio of MC3T3-E1 cells on a titanium disc in a time-dependent manner, the addition of AICAR, which is an AMPK activator, slightly upregulating them on a titanium disc.Moreover, AICAR also upregulated the BMPinduced Id transcriptional activity and the expression of osteogenesis-associated molecules in MC3T3-E1 cells via Smad1/5 phosphorylation on the titanium disc as well as TCPD.Furthermore, BMP-2 in the presence of AICAR induced the expression of their osteoblast differentiationassociated molecules in MC3T3-E1 cells cultured on the titanium disc, to a higher degree than that observed on (unpublished personal data).We recently that AICAR promoted mineralization in MC3T3-E cells on a titanium disc [8].In line with the results of the present study, those results indicated that AMPK activation dominantly enhanced osteoblast differentiation in MC3T3-E1 cells on a titanium disc as well as previous reported on TCPD, suggesting that AICAR synergistically enhances BMP-induced osteoblast differentiation via the Smad1/5 signaling pathway, at least in part.
Autophagy has been reported to play necessary roles in the homeostasis of bone tissue [22].Interestingly, numerous undegraded autophagic vacuoles accumulated in the presence of a halted autophagic flux in undifferentiated mesenchymal stem cells [23], suggesting that these accumulated autophagic vacuoles may serve as a source of rapidly produced energy substrates to support MSC differentiation [14,24].Osteoblasts are specialized mesenchymal-derived cells that are involved in bone formation; in turn, impaired autophagy in osteoblasts leads to a decreased bone mass [14,25,26].
The activation of AMPK, including that observed here, induced autophagy and osteoblast differentiation form stromal/stem cells in bone marrows and periodontal ligaments [6,13,27].In the present experiments, BMP-2 induced the expression of molecules, such as Beclin-1, BNIP3, and LC3-II, in MC3T3-E1 cells on a titanium disc as well as on TCPD.Furthermore, we found that AICAR, which is an AMPK activator, in the presence of BMP-2 also upregulated the expression of autophagy-associated molecules, especially LC3-II, on the titanium disc.In contrast, 3-MA, which is a PI3K inhibitor, suppressed the expression of autophagy-associated molecules, as well as osteoblast differentiationassociated molecules.Although OIM has been reported to stimulate osteoblast differentiation from mesenchymal stromal cells on TCPD via partially AMPK activation BMP-2 had little effect on AMPK phosphorylation on titanium disc.In present experiments, AMPK activation enhanced osteoblast differentiation from MC3T3-E1 cells on a titanium disc via the activation of autophagy.These results indicated that AMPK activated autophagy, resulting in enhancement of osteoblast differentiation in MC3T3-E1 cells pre-osteoblast on a titanium disc.
AdipoRon (APN) has been reported to bind to endogenous adiponectin type 1/type 2 receptors to improve insulin resistance and glucose intolerance [35].APN promoted new bone formation in patients with diabetes mediated by impaired endochondral ossification-induced delayed bone repair in rodent models of diabetes [16,36].The property of APN also induced the mobilization and recruitment of bone-marrow-derived mesenchymal stem cells, to participate in tissue repair [37,38].However, little is known about the mechanism of APN in osseointegration in rodents treated with dental implants in vitro.
In the present study, we found that APN activated AMPK on titanium disc as well as TCPD and then increased the expression of both osteoblast differentiation and autophagy-associated molecules in osteoblasts, suggesting the enhancing effect of AMPK activators on osteoblast differentiation during osseointegration on titanium discs in vitro.
The percentage of failure in the early phase after dental implantation has recently been reported to be higher than that of the late phase in patients with type II diabetes [39,40].Patients with type II diabetes have been reported to exhibit a decrease in the concentration of adiponectin in the serum [41,42].In a histological study, the tissues surrounding the dental implant were observed to be immature and less-organized bone in model animals of uncontrolled diabetes [43].Furthermore, the quality of new bone and osseointegration were significantly reduced, even though insulin-controlled model rats for diabetes [44][45][46].Therefore, the lower level of osseointegration in patients with diabetes is a clinically important event that warrants improvement during dental implant therapy.However, little is known whether AMPK activation using its activator enhance earlier and stronger bone-to-implant contact and osseointegration in vivo, even though diabetes model rodents.Further studies are required to clarify these issues.

Conclusion
In the present study, we found that APN and downstream AMPK signaling enhanced osteoblast differentiation.Furthermore, the upregulation was regulated by autophagy signaling.These results suggest the enhancement of osteoblast differentiation and mineralization in peripheral alveolar bone after implantation, even in patients with diabetes, leading to an early gain of fine osseointegration and implant stability.

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Egashira et al.International Journal of Implant Dentistry (2024) 10:2 Fig.2AICAR enhanced the BMP-2-dependent transcriptional activity on Ti via the expression of osteoblast differentiation-related molecules.A Id promoter site was fused to luciferase and transfected into MC3T3-E1 cells using Lipofectamine 3000.The cells were treated with BMP-2 (50 ng/ mL) with or without AICAR (500 μM) for the indicated times during culture, and luciferase activity was assessed as a measure of Id transcriptional activity.The data are the mean from four culture wells (mean ± SEM).**Indicates P < 0.01 vs. treatment with BMP-2 alone.B-D MC3T3-E1 cells were incubated with BMP-2 in the presence or absence of AICAR (500 μM).Expression of the osteoblast differentiation-related genes Runx2 (Runx2), Osx/Sp7 (OSX/Sp7), and Ocn (Osteocalcin) in MC3T3-E1 cells after incubation with BMP-2 (50 ng/mL).Samples were analyzed by quantitative reverse transcription polymerase chain reaction and were normalized to the ß-actin mRNA.The data are the mean from six culture wells (mean ± SEM).**Indicates P < 0.01 vs. treatment with BMP-2 alone.E Cells were treated with BMP-2 (50 ng/mL) in the presence or absence of AICAR (500 μM).Western blotting was conducted using targeted and ß-actin antibodies.Similar results were obtained in four independent experiments

Runx2
Fig.4 3-MA suppressed BMP-2-induced osteoblast differentiation molecules and autophagy-related molecules.A BMP-2 (25 ng/mL)-induced osteoblast differentiation molecules were mediated by autophagy signaling.The cells were incubated with BMP-2 in the presence or absence of 3-MA (5 mM), which is a PI3K inhibitor.B, C The bar graphs show the mean from five samples (mean ± SEM).* and **Indicate P < 0.05 and P < 0.01, respectively, vs. treatment with BMP-2 alone.D Cells were treated with BMP-2 (25 ng/mL) plus AICAR (500 μM) in the presence or absence of 3-MA (5 mM).Western blotting was conducted using targeted and ß-actin antibodies.Similar results were obtained in three independent experiments

Fig. 5
Fig. 5 Adiponectin enhanced osteoblast differentiation via the AMPK pathway.A Id promoter site was fused to luciferase and transfected into MC3T3-E1 cells using Lipofectamine 3000.The cells were treated with AdipoRon (50 μM) with or without AICAR (500 μM) for the indicated times during culture, and luciferase activity was assessed as a measure of Id transcriptional activity.The data are the mean from four culture wells (mean ± SEM).**Indicates P < 0.01 vs. treatment with AdipoRon alone.B MC3T3-E1 cells were incubated with AdipoRon.Expression of the osteoblast differentiation-related genes Runx2, Osx, and Ocn in MC3T3-E1 cells after incubation with AdipoRon.The samples were analyzed by quantitative reverse transcription polymerase chain reaction and were normalized to the ß-actin mRNA.The data are the mean from six culture wells (mean ± SEM).**Indicates P < 0.01 vs. treatment with AdipoRon alone.C Cells were treated with AdipoRon in the presence or absence of AICAR (500 μM).Western blotting was conducted using targeted and β-actin antibodies.Similar results were obtained in four independent experiments